Correction: Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

[This corrects the article DOI: 10.1371/journal.pbio.

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing.

A Small-Molecule Anti-secretagogue of PCSK9 Targets the 80S Ribosome to Inhibit PCSK9 Protein Translation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that downregulates low-density lipoprotein (LDL) receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C). Phenotypic screening of a small-molecule compound collection was used to identify an inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), which was shown to stimulate uptake of LDL-C in hepatoma cells by increasing LDL-R levels, without altering levels of secreted transferrin. Systematic investigation of the mode of action revealed that R-IMPP did not decrease PCSK9 transcription or increase PCSK9 degradation, but instead caused transcript-dependent inhibition of PCSK9 translation.

Dissociation of ribosomes into large and small subunits.

Structural and functional studies of ribosomal subunits require the dissociation of intact ribosomes into individual small and large ribosomal subunits. The dissociation of the prokaryotic 70S ribosomes into the 50S and 30S subunits is achieved by dialysis against a buffer containing a lower Mg(2+) concentration. Eukaryotic 80S ribosomes are dissociated into 60S and 40S subunits by incubation in a buffer containing puromycin and higher KCl and Mg(2+) concentrations.

Isolation of ribosomes by chromatography.

Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g.

Purification of polysomes.

Isolated polysomes (also known as translating ribosomes or polyribosomes) are mRNA-ribosome complexes that are frequently used for the in vitro study of the regulation of protein synthesis. Here we describe a protocol for the isolation of prokaryotic and plant polysomes by sucrose gradient sedimentation. The protocol allows for the separation of multiple ribosomes attached to mRNA from run-off ribosome monomers.

Purification of 70S ribosomes.

Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.

Isolation of ribosomes and polysomes.

Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts.

Novel 3-O-carbamoyl erythromycin A derivatives (carbamolides) with activity against resistant staphylococcal and streptococcal isolates.

A novel series of 3-O-carbamoyl erythromycin A derived analogs, labeled carbamolides, with activity versus resistant bacterial isolates of staphylococci (including macrolide and oxazolidinone resistant strains) and streptococci are reported. An (R)-2-aryl substituent on a pyrrolidine carbamate appeared to be critical for achieving potency against resistant strains. Crystal structures showed a distinct aromatic interaction between the (R)-2-aryl (3-pyridyl for 4d) substituent on the pyrrolidine and G2484 (G2505, Escherichia coli) of the Deinococcus radiodurans 50S ribosome (3.

A systematic study of 50S ribosomal subunit purification enabling robust crystallization.

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.

Purification of the large ribosomal subunit via its association with the small subunit.

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.

Inhibition of bacterial ribosome assembly: a suitable drug target?

The assembly of bacterial ribosomes is viewed with increasing interest as a potential target for new antibiotics. The in vivo synthesis and assembly of ribosomes are briefly reviewed here, highlighting the many ways in which assembly can be perturbed. The process is compared with the model in vitro process from which much of our knowledge is derived.

A novel chromatography system to isolate active ribosomes from pathogenic bacteria.

We have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Ribosomes interact with an alkyl linker attached to the resin, apparently through their RNA component. Examples are given with ribosomes from Escherichia coli, Deinococcus radiodurans, and with clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA).

A protein component at the heart of an RNA machine: the importance of protein l27 for the function of the bacterial ribosome.

Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid.